Oxidative damage and redox signaling are important components of oncogenic cell transformation, yet the molecular details of how these phenomena impact cellular proteins remain largely uncharacterized. The goal of this proposal is to develop experimental and predictive methods for the identification and molecular analysis of proteins undergoing oxidative modifications at cysteine residues, impacting cancer-related redox signaling pathways in cells. With these efforts, we will be able to apply our new proteomics-based technology to the in situ identification of redox-sensitive signaling proteins in a multi-protein, multi-pathway, whole cell format for the first time. This will allow for investigations to proceed in both discovery- and hypothesis-driven modes to unravel the molecular details of cell signaling pathways responsive to the production of reactive oxygen species (ROS). Multiple studies have highlighted the importance of activated (low pKa) cysteinyl residues in proteins as the primary targets of oxidative modifications, and have shown that hydrogen peroxide is the most important ROS involved in receptor-stimulated cell signaling. The immediate product of peroxide-linked cysteine oxidation is cysteine sulfenic acid (Cys-SOH). Therefore, trapping of Cys-SOH upon its formation in cellular proteins using a detectable reagent will yield a sensitive and comprehensive way of locating redox-responsive cysteinyl residues in cell signaling pathways. To date, no methods exist to trap this species in a manner that is amenable to proteomics type approaches; thus, identification of Cys-SOH containing proteins must be done on an individual, targeted basis. Therefore, we propose four specific aims to (i) create and validate fluorophore- and biotin-linked reagents reactive toward Cys-SOH based on the known sulfenic acid reagent dimedone, (ii) develop "redox-profiling" protocols, using dimedone and reagents created in Aim 1, to trap Cys-SOH in proteins as formed within cells and detect the extent and location of Cys-SOH formation in proteins involved in a particular signaling pathway, (iii) develop "active site profiling" methods using bioinformatics approaches to identify particular cysteinyl residues likely to be targets of redox modifications, and (iv) validate and apply the experimental and predictive methodologies of Specific Aims 2 and 3 as tools in a total cell protein/proteomics format to analyze protein modification during the course of cancer-relevant cell signaling events. These tools will usher in a new era of redox proteomics and enable proteome-scale studies of effects of oxidative stress and antioxidant therapies on cell pathways and signaling networks. [unreadable] [unreadable] [unreadable]